Isobutyrylshikonin has a potentially stronger cytotoxic effect in oral cancer cells than its analogue shikonin in vitro.

OBJECTIVES

The aim of the present study was to identify the anticancer effects and the mechanisms of action of shikonin and its analogue isobutyrylshikonin in oral squamous carcinoma cells.

DESIGNS

The cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22 and SCC-25 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis of Annexin V/Propidium Iodide (PI) staining, western blot analysis and immunohistochemistry.

RESULTS

Treatment with both isobutyrylshikonin and shikonin induced dose- and time-dependent apoptotic cell death in Ca9-22 cells, although the IC of isobutyrylshikonin was less than that of shikonin. The induction of apoptosis by both isobutyrylshikonin and shikonin was accompanied by activation of caspase-8, -9, -3, and PARP, loss of mitochondrial trans-membrane potential, and release of cytochrome c from the mitochondria. ROS mediated the apoptosis induced by isobutyrylshikonin and shikonin, indicating that ROS may play a critical role in the distinctive cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22. Isobutyrylshikonin showed a similar cytotoxic effect in SCC-25 cells at concentrations showing the effects in Ca9 cells, but not in human normal keratinocyte cells. Although there is no biological difference between isobutyrylshikonin and shikonin, isobutyrylshikonin exerts the same cytotoxic effect at a concentration 6 times lower than shikonin.

CONCLUSIONS

The present study suggest that isobutyrylshikonin may be a more potent chemotherapeutic agent against oral cancer cells than shikonin. In addition, our data exhibit that both isobutyrylshikonin and shikonin induce caspase-dependent apoptosis via the mitochondrial pathway through accumulation of ROS in oral squamous carcinoma cells.